Synthetic fusion protein design and applications.

Biomolecular engineering can be used to purposefully manipulate biomolecules, such as peptides, proteins, nucleic acids and lipids, within the framework of the relations among their structures, functions and properties, as well as their applicability to such areas as developing novel biomaterials, biosensing, bioimaging, and clinical diagnostics and therapeutics. Nanotechnology can also be used to design and tune the sizes, shapes, properties and functionality of nanomaterials. As such, there are considerable overlaps between nanotechnology and biomolecular engineering, in that both are concerned with the structure and behavior of materials on the nanometer scale or smaller. Therefore, in combination with nanotechnology, biomolecular engineering is expected to open up new fields of nanobio/bionanotechnology and to contribute to the development of novel nanobiomaterials, nanobiodevices and nanobiosystems. This review highlights recent studies using engineered biological molecules (e.g., oligonucleotides, peptides, proteins, enzymes, polysaccharides, lipids, biological cofactors and ligands) combined with functional nanomaterials in nanobio/bionanotechnology applications, including therapeutics, diagnostics, biosensing, bioanalysis and biocatalysts. Furthermore, this review focuses on five areas of recent advances in biomolecular engineering: (a) nucleic acid engineering, (b) gene engineering, (c) protein engineering, (d) chemical and enzymatic conjugation technologies, and (e) linker engineering. Precisely engineered nanobiomaterials, nanobiodevices and nanobiosystems are anticipated to emerge as next-generation platforms for bioelectronics, biosensors, biocatalysts, molecular imaging modalities, biological actuators, and biomedical applications.

/pdf/biomolecular-engineering-for-nanobio-bionanotechnology-3ddawvh3sy.pdf

Biomolecular engineering for nanobio/bionanotechnology

This review covers the current state of knowledge regarding artificial fusion constructs of cytochrome P450 enzymes in which the activity of the catalytic heme is driven by reductases of different origins. Cytochromes P450 form a vast family of heme-thiolate proteins, which act as monooxygenases by activating molecular oxygen, resulting in the insertion of one atom into an organic substrate with the concomitant reduction of the other to water. The reducing equivalents are usually supplied by nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate and are transferred in two consecutive steps via the redox partner(s). These include reductases containing flavin mononucleotide and/or flavin adenine dinucleotide and/or Fe-S clusters in different combinations depending on the P450 system. These enzymes catalyze extremely diverse reactions, including regio- and stereospecific oxidations of a large range of substrates in addition to many drugs and xenobiotics, as well as biosynthesis of physiologically important compounds such as various steroids, vitamins, and lipids. Because of their ability to catalyze such a vast range of reactions, they have become the focus of biotechnological interest, but their dependence on the reductase partner has remained one of the challenging limitations for full exploration of their synthetic potential. To address the latter limitation, many researchers have reconstituted functional P450 enzymes by fusion with different reductase proteins; this review will cover their findings.

Chimeric P450 enzymes: activity of artificial redox fusions driven by different reductases for biotechnological applications.

Foreword.- 1. Monooxygenase, Peroxidase and Peroxygenase Properties and Reaction Mechanisms of Cytochrome P450 Enzymes .- 2. Oxidizing Intermediates in P450 Catalysis: A Case for Multiple Oxidants.- 3. Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions.- 4. Acyl-Carbon Bond Cleaving Cytochrome P450 Enzymes: CYP17A1, CYP19A1 and CYP51A1.- 5. Regioselective Versatility of Monooxygenase Reactions Catalyzed by CYP2B6 and CYP3A4: Examples With Single Substrates.- 6. Cytochrome P450 Enzymes in the Bioactivation of Polyunsaturated Fatty Acids and their Role in Cardiovascular Disease.- 7. Monooxygenation of Small Hydrocarbons Catalyzed by Bacterial Cytochrome P450s.- 8. Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C-H Bonds of Substrates,- 9. Cytochrome P450 Enzymes and Electrochemistry: Crosstalk with Electrodes as Redox Partners and Electron Sources.- 10. Mechanistic Basis of Electron Transfer to Cytochromes P450 by Natural Redox Partners and Artificial Donor Constructs .- 11. Biological Diversity of Cytochrome P450 Redox Partner Proteins .- 12. Cytochrome P450cin (CYP176A1).- 13. Fungal Unspecific Peroxygenases: Heme-Thiolate Proteins that Combine Peroxidase and Cytochrome P450 Properties.

Monooxygenase, peroxidase and peroxygenase properties and mechanisms of cytochrome P450

In order to elucidate the effect of flexible linker length on the catalytic efficiency of fusion proteins, two short flexible peptide linkers of various lengths were fused between Arabidopsis thaliana 4-coumaroyl-CoA ligase (4CL) and Polygonum cuspidatum stilbene synthase (STS) to generate fusion proteins 4CL-(GSG)n-STS (n ≤ 5) and 4CL-(GGGGS)n-STS (n ≤ 4). The fusion proteins were expressed in both Escherichia coli and Saccharomyces cerevisiae, and their bioactivities were tested in vitro and in vivo using purified proteins and engineered strains, respectively. The catalytic efficiency of the fusions decreased gradually with the increase of GSG or GGGGS repeats. In both engineered S. cerevisiae and E. coli in vivo experiments, the capacity of resveratrol production decreased gradually with increasing linker length. In silico analysis showed that the prediction of homology models of fusion proteins was consistent with the in vitro and in vivo results.

Effect of flexible linker length on the activity of fusion protein 4-coumaroyl-CoA ligase::stilbene synthase.

CYP51, a sterol 14α-demethylase, is one of the key enzymes involved in sterol biosynthesis and requires electrons transferred from its redox partners. A unique CYP51 from Nocardia farcinica IFM10152 forms a distinct cluster with iron-sulfur containing NADPH-P450 reductase (FprD) downstream of CYP51. Previously, sequence alignment of nine reductases from N. farcinica revealed that FprC, FprD, and FprH have an additional sequence at their N-termini that has very high identity with iron-sulfur clustered ferredoxin G (FdxG). To construct an artificial self-sufficient cytochrome P450 monooxygenase (CYP) with only FprD, CYP51, and iron-sulfur containing FprD were fused together with designed linker sequences. CYP51-FprD fusion enzymes showed distinct spectral properties of both flavoprotein and CYP. CYP51-FprD F1 and F2 in recombinant Escherichia coli BL21(DE3) catalyzed demethylation of lanosterol more efficiently, with k(cat) /K(m) values of 96.91 and 105.79 nmol/min/nmol, respectively, which are about 35-fold higher compared to those of CYP51 and FprD alone.

Hyung‐yun Park

Papers

Novel iron-sulfur containing NADPH-reductase from Nocardia farcinica IFM10152 and fusion construction with CYP51 lanosterol demethylase.